In antigen presenting cells, class II molecules of the major histocompatibility complex (MHC) form complexes with peptides derived from endocytosed proteins. Class II molecules of the major histocompatibility complex (MHC) are heterodimeric (.alpha./.beta.), transmembrane glycoproteins expressed by antigen presenting cells (APC) of the immune system (e.g., macrophages, dendritic cells, B cells). Within APC, class II molecules bind peptides derived from endocytosed proteins and from other sources, and the MHC class II/peptide complex is expressed at the cell surface for surveillance by CD4+ T cells. Recognition by the T cell receptor of an antigenic MHC/peptide complex activates the T cell and initiates an immune response.
In human B-lymphoblastoid cell lines, the primary pathway for generating class II molecule/peptide complexes is intimately related to class II molecule biosynthesis. In the endoplasmic reticulum (ER), nascent class II heterodimers associate with the invariant chain (Ii), a class II molecule chaperon; this association promotes correct folding of class II molecules and prevents class II molecule acquisition of peptides in the ER. A targeting or retention signal within the cytoplasmic tail of invariant chain mediates localization of the class II/Ii complex to the endocytic pathway. After cleavage and dissociation of invariant chain, class II molecules bind peptides available in the endocytic pathway, and then traffick to the cell surface.
It had been postulated that the invariant chain may not be the only molecule functioning in this capacity. The requirement for another molecule in this pathway in addition to invariant chain can be inferred from data obtained with mutant human B lymphoblastoid cell lines (B-LCLs). A key feature of these mutants is a defect in presentation in intact protein antigen, but not immunogenic peptides, to class II restricted T cells. In addition, class II molecules expressed by the mutants fail to acquire features of the mature, peptide-loaded molecules of the progenitor cell, such as expression of certain antibody epitopes (e.g., mAb 16.23) and stability in SDS detergent solutions. (Neefjes, et al., EMBO J. (1992) 11:411-416; Mellins, et al., Nature (1990) 343:71-74; and Germain, et al., Nature (1991) 353:134-139, all incorporated herein by reference.) These observations suggested that the mutants are impaired in formation of complexes between class II molecules and peptides derived from endocytosed proteins.
This conclusion regarding the B-LCL mutants is confirmed by direct assessment of the peptides found in association with HLA-DR molecules--human molecules homologous to mouse class II molecules--in the mutants. Rather than the heterogeneous mixture of peptides found in wild type cells, the majority (60-70%) of the DR molecules expressed by the mutants are associated with a nested set of peptides from residues 80-103 of the invariant chain. (Mellins, et al., J. Exp. Med. (in the press); Riberdy, et al., Nature (1992) 360:474-477 and Sette, et al., Science (1992) 258:1801-1804, all incorporated herein by reference.) Though undetectable in the progenitor cell, these Ii peptides have been found in association with class II molecules in other wild type cells (Hunt, et al., Science (1992) 256:1817-1820; Chicz, et al., Nature (1992) 358:764-768 and Chicz, et al., J. Exp. Med. (1993) 178:27-47, all incorporated herein by reference), raising the possibility that the class II/Ii peptide complex is a biosynthetic intermediate. The mutant phenotype thus suggested a number of possible functions for the gene product altered in these cells, including generation or transport of peptides, chaperoning of class II molecules to peptide-containing compartments, or a function that directly facilitates peptide loading of class II molecules, such as removal of Ii peptides.
Although the specific role of its product has not been identified, the relevant gene has been mapped by the present inventor and is reported upon here. The derivation of the mutants from a progenitor cell that is hemizygous for the class II region of the MHC (see 8.1.6, FIG. 1 and Levine, et al., Proc. Natl. Acad. Sci. U.S.A., (1985) 88:3741-3745, incorporated herein by reference) suggested that the responsible gene(s) mapped to this region. B-LCL mutants with homozygous deletions of the region, including the 8.1.6-derived mutant 5.2.4 (FIG. 1 and Mellins, et al., J. Exp. Med. (1991) 174:1607-1615, incorporated herein by reference), were found to share the class II presentation defect, confirming location of the relevant gene(s) within the MHC (Mellins, et al., (1991), supra and Ceman, et al., J. Immun. (1992) 149:754-761, incorporated herein by reference). The gene that underlies the mutant phenotype has been identified as HLA-DMB, an human MHC-linked gene that encodes a class II-like .beta. chain. The results reported below establish HLA-DMB as a critical regulatory molecule in class II restricted antigen presentation and suggest that it functions at an intracellular site to promote class II molecule/peptide association. Transfection of mutants with DMB complementary DNA restored the wild type phenotype, establishing HLA-DM as a critical molecule in antigen presentation to CD4+ T cells.